Blood glucose reacts continuously with proteins in the circulation of human beings. In particular human beings such glucose may bind to amino groups of protein to form aldimines, Schiff bases, which undergo molecular (Amadori) rearrangement to form stable ketosamines.
The various plasma protein-ketosamine products are known as "fructosamines". This reaction occurs predominantly with albumin present in the plasma. Glycation occurs principally (33%) at the lysine residue at position 52 of the albumin. Other sites of glycation include Lys-199, Lys-281, and Lys-439 and there is evidence to support glycation occurring at Lys-12, Lys-233, Lys-317, Lys-351 and Lys-534. Glycation alters the conformation of albumin, as determined by fluorescence studies of the sole tryptophan residue at position 214, and influences the binding properties of albumin. Binding of fatty acids, hemin, bilirubin, and certain drugs is diminished. Reaction also occurs with hemoglobin inside red blood cells to form glycosylated hemoglobin (e.g., hemoglobin Alc). This glycation is believed to occur at the terminal valine residue of the beta chain.
When blood glucose is in proper hormonal balance, glycated protein formation is minimal. However, when blood glucose concentration is elevated for significant periods due to lack of appropriate hormonal control, as in a diabetic patient, then glycated protein concentration, measured as fructosamine concentration in plasma or hemoglobin Alc in blood is elevated above 2 mmol/l and 8%, respectively. Plasma albumin has a metabolic half-life of seventeen days, thus measurement of fructosamine concentration in serum or plasma provides a retrospective record of blood glucose concentrations during the preceding 7-21 days. The metabolic half-life of hemoglobin, packaged inside a red blood cell, is 60-90 days. Thus, testing the amount of glycated hemoglobin provides a retrospective record of control or compliance during the previous 60-90 days. Individually these tests provide different, more relevant, clinical information than a blood glucose measurement which only reflects the current status of glucose metabolism.
A number of tests have been devised to determine the concentration of fructosamines in blood. U.S. Pat. No. 4,200,435 issued Apr. 29, 1980 to Stroupe and U.S. Pat. No. 4,255,385 issued Mar. 10, 1981 to Stroupe disclose methods, test kits and reagents for detecting glycosylated hemoglobin in blood by measuring the change in distribution of the allosteric forms of hemoglobin in blood samples. U.S. Pat. No. 4, 247,533 issued Jan. 27, 1981 to Cerami et al., discloses antibodies specific for glycohemoglobin Alc for use in immunoassays for detecting fructosamines in blood. U.S. Pat. No. 4,269,605 issued May 26, 1981 to Dean et al., discloses methods of separating glycoproteins from non-glycosylated proteins in a mixture by binding the glycoproteins to a dihydroxyboryl group bonded to a support. Generally, this takes the form of passing a blood sample through a column containing the reactive agent where glycoproteins bind to the dihydroboronyl group. Non-binding components blood are washed away, and the glycoproteins are then eluted from the column.
In the method disclosed in U.S. Pat. No. 4,349,352 issued Sep. 14, 1982 to Manning, isolated glycoprotein is treated with a phenylhydrazine and the absorption coefficient of the resulting phenylhydrazine is then measured. U.S. Pat. No. 4,399,227 issued Aug. 16, 1983 to Niederau, discloses a method of reducing interference by unstable glucose-aldimine-hemoglobin compounds with an assay for determining glycosylated hemoglobin. These tests, however, can be time-consuming and labor-intensive to perform. Persons suffering from diabetes must have their blood glucose levels checked regularly to assure the proper efficacy of their treatment. Time-consuming tests that cannot be performed in the physician's office while the patient is present are inconvenient and increase the likelihood of patient non-compliance with the prescribed treatment.
U.S. Pat. No. 4,642,295 issued Feb. 10, 1987 to Baker and U.S. Pat. No. 4,645,742 issued Feb. 24, 1987 to Baker disclose tests and reagents for determining fructosamine levels in blood by reacting a blood sample with a coloring agent, taking a first measurement of light absorption and at a suitable later time, taking a second light absorption measurement, and comparing any change in measurements, such that any change in color between the first and second measurement is caused predominantly by glucose in the sample that is reacted or associated with an amine group of protein and has undergone a molecular rearrangement to form fructosamine. This test is relatively inexpensive and simple to perform, but is subject to a certain amount of interference by other components of blood.
There is thus a need for rapid, reliable tests for detection of fructosamines in blood that can be performed by relatively unskilled personnel without the need for sophisticated laboratory equipment. Accordingly, it is an object of the invention to provide such tests for measuring levels of fructosamines in blood.